Microtubule-based machines are responsible for the determination of cell shape, intracellular transport of organelles, chromosome separation during mitosis, and beating of cilia and flagella. Defects in the function of the motor proteins that drive these machines result in changes in cell shape, misplacement of organelles, infertility, respiratory disease, and other developmental defects. The long-term goal of the proposed research is to understand the mechanisms that regulate the assembly, targeting, and activity of the dynein family of motors. We have identified several new genes that are involved in the assembly and regulation of dynein motors in Chlamydomonas. We will continue to capitalize on the highly ordered structural organization of the flagellar axoneme and the ease of genetic analysis in Chlamydomonas to further characterize these genes and gene products and identify interacting components that regulate dynein activity. Our specific aims are: 1) To characterize a novel gene that encodes a dynein intermediate chain (IC138) involved in the regulation of the l1 inner arm dynein. We have identified an unusual motility mutant that lacks the IC138 regulatory subunit, but still assembles the l1 complex into the axoneme. We will test the hypothesis that the loss of IC138 inhibits l1 activity using in vitro assays and high resolution structural methods to analyze subunit interactions within the complex. (2) To identify and characterize interactions between components of a dynein regulatory complex (DRC). We will test the hypothesis that the DRC alters doublet sliding velocities by modulating the activity of axonemal kinases and phosphatases. We have identified the first DRC subunit as a novel but highly conserved protein. Epitope-tagged constructs and specific antibody probes will be used to further define the biochemical properties of the DRC. Biochemical methods and genomic strategies will be used to identify other components of the DRC. (3) To identify components that interact with a cytoplasmic dynein required for retrograde intraflagellar transport (IFT). Biochemical and genomic strategies will be used to identify new subunits and trans acting regulators of the cDhd b complex. The studies will provide basic information about the organization of dyneins and associated regulatory components in the axoneme and new insights into the mechanisms that target the dyneins to specific locations and regulate their activities. Given the critical roles played by motor proteins and cilia and flagella in a wide range of human diseases, the studies will also have important implications for diagnostic and therapeutic strategies in the treatment of human disease.